1. Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases
- Author
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Erik J. de Vries, Dick B. Janssen, Esther M. Gabor, Biotechnologie, Faculty of Science and Engineering, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Geologic Sediments ,Genetic Vectors ,Molecular Sequence Data ,Glycine ,DIVERSITY ,Penicillin amidase ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Microbiology ,Insert (molecular biology) ,PENICILLIN ACYLASE ,Amidohydrolases ,law.invention ,Amidase ,chemistry.chemical_compound ,law ,GRADIENT GEL-ELECTROPHORESIS ,Acetamides ,Escherichia coli ,Amidase activity ,medicine ,Environmental DNA ,MICROORGANISMS ,GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries) ,Chromatography, High Pressure Liquid ,Soil Microbiology ,Ecology, Evolution, Behavior and Systematics ,DNA Primers ,Gene Library ,Netherlands ,Base Sequence ,Sequence Analysis, DNA ,LIBRARIES ,METAGENOME ,Gene Components ,chemistry ,Biochemistry ,ESCHERICHIA-COLI ,DISCOVERY ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,Recombinant DNA ,BETA-LACTAM ANTIBIOTICS ,ENVIRONMENTAL DNA ,DNA ,Plasmids - Abstract
Summary To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.
- Published
- 2004